Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1′681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.


Introduction
The larval metacestode stage of the small fox tapeworm Echinococcus multilocularis causes the disease alveolar echinococcosis (AE). The zoonosis involves carnivores as definitive hosts and a diversity of small mammalian species (mostly rodents) as intermediate hosts. Humans, monkeys and other mammals can be affected as aberrant hosts when infected with eggs from E. multilocularis (Lymbery et al., 2017). The resulting disease AE is characterized by the clustered, tumor-like growth of metacestodes mainly in the liver of patients. The course of the disease is asymptomatic in the first years of infection, but when untreated, AE becomes progressive and life-threatening. E. multilocularis ranks first and third in the respective European and global rankings of food-borne parasites (Bouwknegt et al., 2018). This is due to the fact that AE is fatal in untreated patients, and curative drug treatment lacks to date (Lundström-Stadelmann et al., 2020). Treatment options rely on complete surgical resections performed in 20-50% of infected people in countries where healthcare infrastructure is easily accessible . Following surgery, patients are recommended for chemotherapeutic treatment with the benzimidazoles (BMZs) albendazole or mebendazole for at least two years, and for regular monitoring for at least ten years (Brunetti et al., 2010). Unfortunately, AE is usually diagnosed at a late, already advanced stage, making radical surgical treatment difficult or impossible. Then the only treatment option left is based on BMZ treatment. Chemotherapeutic treatment with BMZ is limited by parasitostatic effects and therefore requires long-term (often life-long) treatment (Lundström-Stadelmann et al., 2020). BMZ are tolerated in 70-80% of AE patients, with elevated transaminases, proteinuria, transient hair loss, gastrointestinal disturbances, leukopenia and neurological symptoms as the most common adverse effects . In a German long-term observational study, 6.9% suffered from severe liver toxicity (Grüner et al., 2017). Thus, there is an urgent need for new drugs to successfully treat AE.
In a drug-repurposing approach, the anti-malarial mefloquine (MEF) was used against E. multilocularis in vitro and in infected mice and in these studies MEF exhibited promising, albeit not parasiticidal, activity (Küster et al., 2011(Küster et al., , 2015Lundström-Stadelmann et al., 2020). When used in malaria prophylaxis, mefloquine can induce strong neuropsychiatric side-effects (Tickell-Painter et al., 2017). A first clinical report of an AE patient that was treated with MEF did not lead to a positive treatment response, but no adverse effects did occur either (Burkert et al., 2022). This further confirms the potential lack of parasiticidal activity of MEF in an in vivo setting. Comparative in vitro screening of MEF derivatives was performed against E. multilocularis metacestodes, but none of the tested compounds showed improved activity compared with MEF (Rufener et al., 2018).
Though several targets are described for MEF in Plasmodium, bacteria and mammals , the molecular targets of MEF in E. multilocularis are unknown. Former investigations using affinity chromatography revealed enolase as a major MEF-binding protein in Schistosoma mansoni (Manneck et al., 2012), ferritin in E. multilocularis (Küster et al., 2015), and nicotinamide phosphoribosyltransferase in human cells (Küster et al., 2015). In these studies, the major binding proteins were identified after SDS-PAGE of eluates followed by staining and excision of the most visible bands, which may have caused a bias. Moreover, appropriate controls, i.e. pull-downs with ineffective MEF derivatives, were lacking. Therefore, in order to get up-to-date insight into molecular targets of MEF, another methodology was used in this study. Differential affinity chromatography (DAC) is based on pull-downs of cell free extracts on columns coated with effective compounds or ineffective derivatives followed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) of the eluates. In recent studies, DAC has been used to identify molecular targets of ruthenium complexes in Toxoplasma gondii and Trypanosoma brucei (Anghel et al., 2021), and to identify molecular targets of a bumped kinase inhibitor in Neospora caninum and Danio rerio (Müller et al., 2022a) and proteins binding to Leucinostatin-like peptides (Müller et al., 2022b).
Taking advantage of the investigation of a new series of MEF derivatives followed by a structure activity relationship (SAR) study, we performed DAC with effective and ineffective MEF derivatives in order to identify potential drug targets of MEF in E. multilocularis metacestodes.

Materials and methods
All chemicals were purchased from Merck (Darmstadt, Germany), if not stated otherwise. Cell culture reagents were from Gibco (through Fisher Scientific AG, Reinach, Switzerland) if not indicated differently.

Synthesis and chemical analysis of 14 MEF derivatives
The structures of MEF derivatives are given in Fig. 1. The MEF derivatives ( Fig. 1) were synthesized at Fundação Oswaldo Cruz as previously described (da Silva et al., 2021). MEF-1 to MEF-10 were previously applied in Rufener et al. (2018). MEF-11 to MEF-24 had not previously been tested against E. multilocularis and were tested in this study. In short, the first step was the condensation of substituted anilines with ethyl 4,4,4-trifluoro-3-oxobutanoate in presence of PPA at 150 • C for 2-3 h to induce formation of the substituted quinoline nucleus with a free hydroxyl group at position 4. These phenols were then alkylated with methyl iodide or ethyl bromide in acetone at room temperature in the presence of Na 2 CO 3 for 4-20 h to produce the key intermediates with a 50-70% yield, respectively. These alkylated compounds were subjected to nucleophilic substitution with diamines, alkylamine, and amino alcohols at 90-120 • C to produce the derivatives MEF-3, MEF-7, MEF-9, MEF-10,  with a 15-76% yield after 1-3 h of reaction . The compounds MEF-7, MEF-10 and MEF-14, MEF-22 were converted to the chloro-derivatives MEF-6, MEF-8 and MEF-15, MEF-23 by treatment with SOCl 2 in CH 2 Cl 2 or CHCl 3 at reflux with a 75-95% yield after 1-3 h of reaction. The compounds MEF-11, MEF-20 were obtained after reaction of MEF-6, MEF-8 with ethanolamine under heating at 110 • C for 1-2 h with a 64-79% yield. The azido compounds MEF-12, MEF-21 were obtained after reaction of MEF-6, MEF-8 with sodium azide in DMF at 130 • C for 2-3:30 h in 56-79% yield. The compounds MEF-16, MEF-24 were obtained after reaction of MEF-15, MEF-23 with ethanolamine at 110 • C for 1-2 h in 70-90% yield.
All these compounds were identified by detailed spectral data, including 1 H-NMR, 13 C-NMR, and high resolution mass spectra. In general, the 1 H-NMR spectrum showed four or five quinoline protons at 8.90-6.20 ppm and the aliphatic protons at 4.00-0.95 ppm. The 13 C-NMR spectrum showed the quinoline carbon signals at the region of 93-165 ppm and the aliphatic carbons at the regions of 14-61 ppm. The CF 3 group showed a quartet with J about 270-274 Hz. In the infrared spectra, characteristic signals of NH were observed at 3200 -3380 cm − 1 , OH signals were observed at 3500 -4000 cm − 1 and N 3 signals were observed at 2090 -2100 cm − 1 . For all compounds the C-F axial deformation signals were observed at 1080 -1300 cm − 1 .
Melting points were determined with a MQAPF-302 Micro Química apparatus and are uncorrected. NMR spectra were determined using 400 or 500 MHz Bruker AC spectrometers using tetramethylsylane as internal standard. Splitting patterns are as follows: s, singlet; d, duplet; d, double duplet; t, triplet; quin, quintet; m, multiplet; Brl, broad signal. Infrared spectra were obtained using a Thermo Nicolet 6700 spectrometer. Mass spectra were recorded on Agilent 122 5532 GC/MS column by electron impact and high resolution spectra on Bruker compact-Tof. The progress of the reactions was monitored by thin-layer chromatography (TLC) on 2.0 cm × 6.0 cm aluminium sheets (silica gel 60, HF-254, Merck) with a thickness of 0.25 mm, ultraviolet light irradiation. For column chromatography, Merck silica gel (70-230 or 230-400 mesh) was used. Solvents and reagents were used without further purification.

E. multilocularis strain maintenance in mice
Female BALB/c mice (Charles River, Sulzfeld, Germany) were used for parasite strain maintenance at the Institute of Parasitology in Bern. They had access to food and water ad libitum and were kept in ventilated cages with a controlled temperature of 21 • C-23 • C, a relative humidity of 45%-55%, and a 12/12 h light/dark cycle. Animals were kept under the cantonal license number BE30/19 approved by the Animal Welfare committee of the canton of Bern. All animals were treated in compliance with the Swiss Federal Protection of Animals Act (TSchV, SR455). Intraperitoneally infected BALB/c mice were euthanized after 2-4 months post infection to isolate E. multilocularis metacestodes (isolate H95) as previously described (Rufener et al., 2018).

E. multilocularis metacestode in vitro culture
E. multilocularis metacestodes were cultured as described before at the Institute of Parasitology Bern (Rufener et al., 2018). The metacestode material resected from infected mice was incubated overnight at 4 • C with 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml tetracycline and 20 μg/ml levofloxacin. The parasite pellet was taken up in Dulbecco's Modified Eagle Medium (DMEM) with phenol red, 4.5 g/l D-glucose and pyruvate, supplemented with 10% fetal calf serum (FCS, Biochrom GmbH, Berlin, Germany), 100 U/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml tetracycline. The suspension was transferred to a 75 cm 2 (T75) culture flask with confluent Reuber rat hepatoma (RH) feeder cells. The new co-culture was incubated at 37 • C and 5% CO 2 under humid atmosphere. The medium of the co-culture was changed once a week. The RH culture was maintained in the same culture medium and the same conditions as the metacestodes and passaged once a week to be added to the metacestodes.

In vitro drug screening against E. multilocularis metacestodes
14 MEF derivatives (MEF-11 to MEF-24) were tested against E. multilocularis metacestodes by PGI assay, which measures damage of metacestodes via the damage marker phosphoglucose isomerase (PGI) (Stadelmann et al., 2010). Metacestodes of 6-8 weeks of age were used for these in vitro tests. Purified metacestodes were distributed to 48 well plates diluted 1:2 in DMEM (without phenol red, supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin). In a first screen, all 14 MEF derivatives were tested in triplicates at a final concentration of 40 μM in 0.1% dimethyl sulphoxide (DMSO), with an incubation time of 5 days, 5% CO 2 , humid atmosphere. In a second screen, the five most effective MEF derivatives (>20% activity in first screen) were tested at 40 μM, 30 μM, 20 μM and 10 μM, each in triplicate, for 5 days, 5% CO 2 , humid atmosphere. For all assays, respective DMSO (0.1%) and Tx-100 (0.1%) controls were included in triplicates. After 5 days of metacestode incubation, supernatants were collected and stored at -20 • C until measurement. PGI activities were measured indirectly via formation of NADH as described before (Stadelmann et al., 2010). Activities were calculated as percentage of the Tx-100 control. They are given as mean values and standard deviations for each triplicate.

Affinity chromatography of E. multilocularis metacestode proteins to MEF derivatives (a) Coupling of MEF-ligands to Sepharose matrix
The coupling of MEF, MEF-3, MEF-13 and MEF-22 to epoxyactivated sepharose® 6B with a C12 spacer arm was performed according to the manufacturer's instructions as described previously (Müller et al., 2008) with the exception that MEF or derivatives were used as ligands and not thiazolides. In short, 0.5 g of lyophilized epoxy-sepharose® 6B (Sigma-Aldrich, St. Louis, Mo, USA) was suspended in 15 ml H 2 O (Bichsel AG, Interlaken, Switzerland) and centrifuged at 300×g for 5 min at 4 • C. Washing in water was repeated twice, followed by a two-stage wash with coupling buffer (0.1 M Na 2 CO 3 , pH 9.5). 15.5 ± 0.3 mg of the ligand was weighed and dissolved in 1 ml DMSO, added to 1.5 ml of the washed epoxy-sepharose resin and incubated for 96 h at 37 • C on a horizontal shaker with slow but continuous shaking (150 rpm) to allow the drug to react in a nucleophilic addition to the epoxy-activated sepharose® 6B. After 72 h of incubation, 2 ml of DMSO was added to the resin to dissolve all excess and possibly precipitated drug in the supernatant to further increase the coupling efficiency. After incubation, the matrix was washed again with coupling buffer, followed by another wash with ethanolamine (1 M, pH 9.5). The matrix was overlaid with ethanolamine (1 M, pH 9.5) and incubated for 4 h at 20 • C on a horizontal shaker at 150 rpm in the dark to block residual groups. The resulting column medium was then transferred to an empty PD-10 column (GE Healthcare, Glattbrugg, Switzerland) and washed with PBS/DMSO (1:1) (5 column volumes) to remove unbound ligands. The resulting column was stored sealed in PBS containing 0.02% NaN 3 at 4 • C until use. Mock columns were prepared in the same way, but without any ligand.
(b) Metacestode protein extraction for affinity chromatography Extracts were prepared from in vitro cultured E. multilocularis metacestodes maintained under axenic aerobic conditions (5% CO 2 , 20% O 2 , humid atmosphere, 37 • C) without RH cells or FCS and DMEM containing 150 U/ml penicillin and 150 μg/ml streptomycin. After 72 h, the metacestodes were washed extensively in PBS, destroyed after removal of all PBS and centrifuged at 500×g for 5 min at 4 • C. This washing step was performed twice more. To lyse the cells in the tissue pellet, 4 ml of PBS containing Tx-100 (0.1%), 5 mM EDTA and 10 μl/ml Halt Protease Inhibitor cocktail (Thermo Fisher) were added and then vortexed for 1 min. After centrifugation of the suspension at 4000×g for 10 min, 4 • C, 8 ml of supernatant was re-centrifuged in 1.5 ml Eppendorf tubes at 12 ′ 000×g for 10 min at 4 • C. The supernatant corresponding to the cell extract was immediately used for differential affinity chromatography.
(c) Differential affinity chromatography (DAC) Differential affinity chromatography was performed similarly as described previously (Anghel et al., 2021;Müller et al., 2022aMüller et al., , 2022b. Parasite extracts were passed first through a mock column (Küster et al., 2015) followed by a column containing effective or ineffective drugs and at a flow rate of 0.25 ml/min. Prior to elution, the columns were washed with at least 5 column volumes of PBS. Mock and drug columns were eluted with 4 ml of acetic acid (50 mM, pH 2.9) each. The eluates were aliquoted (1 ml) and lyophilized overnight on a Savant SC100 SpeedVac concentrator for subsequent analysis by nano-liquid chromatography coupled tandem mass spectrometry (nLC-MS/MS) as described previously (Müller et al., 2022b).

Mass spectrometry and data processing
nLC-MS/MS was performed to identify the protein composition of the above-described samples (a) and a specific workflow of data processing and visualization (b), protein annotation (c) and Gene Ontology enrichment analysis (d) was used.

(a) nLC-MS/MS
At the Proteomics and Mass Spectrometry Core Facility, University of Bern, eluates were re-suspended in 10 μl lysis buffer (8 M Urea/100 mM Tris-HCl pH8), reduced by addition of 1 μl 0.1 M DTT for 30 min at 37 • C, alkylated with 1 μl of 0.5 M iodoacetamide for 30 min at 37 • C in the dark, followed by quenching of iodoacetamide by the addition of 5 μl 0.1 M DTT and digested with LysC for 2 h at 37 • C followed by Trypsin overnight at room temperature. The digests were analysed by liquid chromatography on an Ultimate 3000 (Thermo Fisher, Bremen, Germany) coupled to a LUMOS tribrid orbitrap mass spectrometer (Thermo Fisher, San José, USA) with two injections of 500 ng peptides. The samples were loaded in random order onto a pre-column (C18 PepMap 100, 5 μm, 100 A, 300 μm i.d. X 5 mm length) at a flow rate of 10 μl/min with solvent C (0.05% TFA in water/acetonitrile 98:2). After loading, peptides were eluted in back flush mode onto a homemade C18 CSH Waters column (1.7 μm, 130 Å, 75 μm × 20 cm) by applying a 90-min gradient of 5% acetonitrile to 40% in water, 0.1% formic acid, at a flow rate of 250 nl/min.
Data acquisition was made in data dependent mode with precursor ion scans recorded in the orbitrap at a resolution of 120 ′ 000 (at m/z = 250) parallel to top speed fragment spectra of the most intense precursor ions in the linear iontrap for a cycle time of 3 s maximum, applying a 30s exclusion time window.

(b) Data processing and visualization
Mass spectrometry data was processed by MaxQuant software (version 1.6.14.0) using default parameter settings with 10 ppm mass accuracy for precursor ion masses in the first search run, and 0.4 Da mass accuracy for fragment spectra recorded in the linear iontrap. A fixed modification of carbamidomethylation on cysteines and variable modifications of protein N-terminal acetylation, oxidation on methionine, and deamidation of asparagine and glutamine were set for a database search against echinococcus_multilocularis.PRJEB122.WBPS15.pro-tein_1.fasta (download date March 9, 2021) sequence database deactivation match between runs option. Proteins at a 1% FDR with at least two razor and unique peptide identifications (also with a 1% FDR setting) were accepted as present in the sample. Subsequent data analysis was performed in Microsoft Excel 2019, applying the data processing workflow as follows (Fig. 2): Only strictly detected lead proteins in both duplicates per drug column and not in the corresponding mock column were characterized. To reduce the number of non-specifically binding proteins, only those proteins that were bound exclusively by the effective drugs MEF and MEF-3 were considered. Finally, hits were sorted by descending iBAQ values, which are the quotients of their intensities and the number of tryptic peptides, to determine the top 25 bound proteins by MEF and MEF-3. For Venn Diagram generation the online tool InteractiVenn was used (Heberle et al., 2015).

(c) Protein Annotation
Annotations for all detected proteins were generated by BlastP of each gene (E. multilocularis genome version WBPS 15, https://parasite. wormbase.org) against the Uniprot Database Eukarya (https://uniprot. org, 2022-10-01) with an e-value of 1e-5. Only top hits for each gene were included for further analyses. Top 25 bound proteins by both MEF and MEF-3 that did not get a hit by this BlastP analysis were analysed individually using the online tool of NCBI conserved domains (Lu et al., 2020). Functional grouping of these proteins was performed based on the information given in Uniprot.

(d) Gene Ontology (GO) enrichment analysis
For the analysis of GO terms two online tools were used: g:Profiler (Raudvere et al., 2019) and Enrichment Analysis in Caenorhabditis elegans (Angeles-Albores et al., 2016. Proteins bound exclusively by the effective drugs MEF and MEF-3 were subjected to this GO enrichment analysis using the respective E. multilocularis proteins as a query against the proteome of C. elegans (genome version WBPS 15, https://parasite.wormbase.org) with an e-value of 1e-5 to retrieve homologues in C. elegans. C. elegans is the only helminth species for which GO enrichment analysis is available to date. For further analyses and GO term enrichment was performed for the top hits of each protein in the three classic categories Cellular Component (CC), Biological Process (BP) and Molecular Function (MF), and the levels 3 and 4 are shown in the results. For each GO term the negative log(10) of the P value was plotted.

Fig. 3. Activity of MEF and derivatives against E. multilocularis metacestodes.
The compounds were tested at 40 μM against E. multilocularis metacestodes for 5 days under standard conditions (5% CO2, 21% O2, humid atmosphere) in biological triplicates. Average PGI values (AV) and standard deviations (SDs) are given in % of the positive control Tx-100 for each triplicate. Effective compounds (>20% PGI release) are indicated by *. For comparison, this table also includes the data published previously by Rufener et al. (2018) for MEF-1 -MEF-10.

MEF derivatives with activities against E. multilocularis metacestodes
In order to identify effective and ineffective MEF derivatives for subsequent analysis of affinoproteomes by DAC, an overview screening of the 14 MEF derivatives (MEF-11 -MEF-24) and MEF at 40 μM was performed to evaluate their activities against E. multilocularis metacestodes. Fig. 3 shows the PGI release relative to the positive control Tx-100 (for comparison, this table also includes the data published previously by Rufener et al. (2018) for MEF-1 -MEF-10). Four new compounds were effective (>20% of Tx-100 activity) in this first screen apart from MEF: MEF-11, MEF-12, MEF-15, and MEF-16. MEF-15 showed the highest activity compared to the other MEF derivatives, with a relative PGI activity of 57.6% (±3.2%) after 5 days of incubation. None of the MEF derivatives showed higher activities than MEF (77.1% ± 0.0%). The other MEF derivatives  were ineffective after 5 days of incubation at 40 μM (Fig. 3).
To confirm the MEF derivatives with activities of 20% or more in the first screen, they were tested in a PGI assay at concentrations of 10, 20, 30 and 40 μM. Fig. 4 confirms that none of the derivatives showed higher activities than MEF, which still exhibited an average relative PGI release of 81.7% (±6.5%) at 20 μM. MEF-16 showed the highest efficacy among the derivatives at 20 μM with an activity of 6.3% (±9.9%). MEF-13 and MEF-22 were also tested at lower concentrations as negative controls. They were again ineffective at all concentrations in these repeated tests.

DAC of MEF binding proteins
To identify potential protein targets of MEF in cell-free extracts of E. multilocularis, DAC was performed with the effective compounds MEF and MEF-3 and the ineffective MEF-13 and MEF-22. These compounds differ by the side chain in residue R 2 and by the CF 3 group in residue R 1 .
A total of 2 ′ 782 binding proteins were identified by this approach (Fig. 5, Supplementary Table S1). Contaminating proteins and proteins only identified by site were omitted from further analysis. Of the remaining 2 ′ 668 proteins (Supplementary Table S2), only those 1 ′ 681 binding proteins that were strictly detected in both duplicates per drug column, and not in the corresponding mock columns (Supplementary  Table S3), were further analysed. Of all 1 ′ 681 proteins specific for MEF or derivatives, 960 proteins were detected in the MEF-coupled column, 917 proteins were detected in the MEF-3-coupled column, 879 proteins detected in the MEF-13-coupled column and 73 proteins were detected in the MEF-22-coupled column (Fig. 5). 269 proteins (16.00%) bound to MEF exclusively (Fig. 5, Supplementary Table S4) and 248 to MEF-3

Discussion
Infection with E. multilocularis larvae causes the disease AE with infiltrative growth in the liver (Stojkovic and Junghanss, 2013). The high fatality rate in untreated patients makes AE one of the most dangerous food-borne parasitoses in the world (Lymbery et al., 2017). New chemotherapeutic treatment options are urgently needed. Drug repurposing is a strategy that might efficiently aid in the identification of alternative drug treatments for AE, as for other neglected diseases (Panic et al., 2014;Lundström-Stadelmann et al., 2020). One of the most important heterocyclic compounds in the field of medicinal and organic chemistry is quinoline, which occurs in the backbone of molecules with great biological importance of natural (Musiol et al., 2017;Nainwal et al., 2019) or synthetic origin (Narwal et al., 2017;Sharma et al., 2017;Shang et al., 2018), such as quinine, camptothecin or skimminanine (Musiol et al., 2017;Nainwal et al., 2019). These bioactive quinoline-based compounds were the precursors of several derivatives that are now used clinically and contribute greatly to the control of serious diseases worldwide (Narwal et al., 2017;Sharma et al., 2017;Shang et al., 2018;Kucharski et al., 2022). In addition, several other compounds with quinoline nuclei fused or unfused to other heterocyclic compounds have been synthesized recently, exhibiting a wide range of biological properties in vitro and in vivo, such as antimalarials (Leven et al., 2019), antifungals (Shruthi et al., 2019), anticancer agents (Hamdy et al., 2019), anticonvulsants (Kumar and Abdullah, 2019), antihypertensives (Vellalacheruvu et al., 2017), anti-inflammatories (Deaton et al., 2019), antidepressants (Galambos et al., 2017), and antivirals (Kos et al., 2019).
For MEF, which has a quinoline nucleus substituted by two trifluoromethyl groups, activity against E. multilocularis metacestodes was demonstrated in recent years (Küster et al., 2011(Küster et al., , 2015Lundström--Stadelmann et al., 2020). Even though the drug is active in vitro at relatively high concentrations, the same holds true for the current drug in use, albendazole. In the murine AE model, mefloquine is the only drug that to date reaches comparable parasite reduction as albendazole. Building on previous studies that evaluated the importance of specific chemical groups for MEF activity, new analogues were evaluated in the present study to determine likely mechanisms of action for MEF and its derivatives. Our in vitro testing of 14 new derivatives of MEF against E. multilocularis metacestodes clearly showed that none of the 14 MEF derivatives showed higher activity compared to MEF in the PGI assay against E. multilocularis metacestodes. This is in contrast to a previous SAR is a very useful to follow structural modifications of a compound to optimize its property or activity (Guha, 2013). In order to find new effective derivatives of MEF, which ideally would be less toxic than MEF, the structures of MEF derivatives were compared in a SAR study. MEF-derivatives 17-24 were found to be ineffective against metacestodes. Their common feature was the lack of a trifluoromethyl group at position 8 (R 1 ) of the quinoline core structure. These findings are consistent with previous results obtained with MEF derivatives (MEF-1 -MEF-10) tested in vitro against E. multilocularis metacestodes (Rufener et al., 2018). In contrast, MEF-13 and MEF-14, which contained a trifluoromethyl group at R 1 and a secondary amine in R 2 , showed no activity against metacestodes. Similar studies by Dassonville-Klimpt et al. (2011) and Barbosa-Lima et al. (2017) showed that derivatives containing trifluoromethyl groups were effective against Plasmodium spp. and secondary amine side chains were effective against the Zika virus. It appears that the inclusion of a chlorine atom in the side chain at position 4 (R 2 ) contributes to the increased activity for MEF-7 versus MEF-8, MEF-14 versus MEF-15, MEF-10 versus MEF-6, MEF-17 versus MEF-8 and MEF-22 versus MEF-23, with the exception of MEF-3 versus MEF-6. Small groups located at position 4 of the ring (R 2 ) provided inactive derivatives, for example MEF-2, MEF-4 and MEF-5. Inclusion of a methylene group in the side chain at the R 2 of MEF-3 resulted in the less active derivative MEF-13. The same was also observed forMEF-10 that lost its activity with an additional methylene group in MEF-14. On the other hand, the insertion of a methylene group in the side chain of MEF-6 resulted in a more active derivative MEF-15, and the same was observed in MEF-11 in relation to MEF-16. MEF-15, which contained three methylene groups, was effective and differed from MEF-13 and MEF-14 in that it contained a chlorine at the end instead of a primary amine or hydroxyl group. The fact that most of the derivatives such as MEF-11, MEF-12, MEF-16 and the first-generation derivatives from Rufener et al. (2018), contained good nucleophiles at the end of the side chain suggests that nucleophiles could play a role in the activity, but could be influenced by the length of the side chain, as in the case with MEF-13 and MEF-14.
Concerning the length of the side chain, another observation was made with MEF-15 and MEF-16. It can be assumed that MEF-16, the most promising second-generation effective derivative against E. multilocularis metacestodes, showed activity due to the similar Table 1 Top 25 proteins from the cell extract bound to both MEF and MEF-3 during affinity chro-matography and identified by nLC-MS/MS. Out of the 216 proteins described as the "MEF specific affinoproteome", the top 25 proteins were determined according to the highest iBAQ average from duplicates of MEF. The columns indicate the protein ID in WormBase ParaSite, the Protein ID and Protein Description of the best BlastP hit in Uniprot, or Conserved domain analysis where BlastP did not give any hits (indicated by *), as well as the iBAQ values for MEF and MEF-3. functional groups with a total of 8 atoms in the long R 2 residue as in the 2-piperidylmethanol group of MEF. Therefore, one idea of a possible interaction of MEF-16 with the target could be in mimicking the 2-piperidylmethanol structure of MEF. An interaction of the 2-piperidylmethanol of MEF with amino acids in a binding pocket was demonstrated in a previous study where MEF bound to acyl-CoA-binding proteins of Plasmodium falciparum . Further research with new derivatives is needed to broaden the SAR study of MEF. The phenotypical screening of MEF derivatives has provided us with one effective (MEF-3) and two ineffective derivatives MEF-13 and MEF-22 with structural similarity to MEF. Consequently, DAC using these compounds was performed in order to identify E. multilocularis proteins specifically binding to MEF or effective MEF derivatives resulting in the identification of 1 ′ 681 proteins. A potential downside of affinity chromatography upon drug-coupling to a solid support lays in that by coupling the potential target-binding affinity can be changed. However, the same basic method of coupling of MEF for affinity chromatography was applied before (Manneck et al., 2012;Küster et al., 2015). Moreover, the low number of exclusively detected proteins bound by MEF-22 (3.51%) and the corresponding high number of exclusively bound proteins by MEF (16.00%) and by MEF-3 (14.75%) were consistent with the expectation regarding protein binding in relation to activity. What was unexpected was the comparatively equal number of proteins bound exclusively to the ineffective MEF-13 (15.65%). Despite the biological inactivity of MEF-13 towards E. multilocularis metacestodes, the protein binding ability could be explained by the fact that the trifluoromethyl group (R 1 residue) contained at position 8, which is not present in the structure of MEF-22, interacts non-specifically with many proteins.
As shown before (Müller et al., 2022a), DAC is a useful tool to identify affinoproteomes specific for effective compounds. In the present study, the use of biologically ineffective analogues of MEF with high similarity to the effective compounds allows to remove non-specifically bound proteins from the affinoproteome of effective MEF-analogues. However, with 216 proteins this specific affinoproteome is still very large.
Since most of these 216 binding proteins are only detectable in small amounts, it is safe to focus on the 25 most abundant proteins bound by both effective compounds MEF and MEF-3. These "Top 25" include proteins involved in pathways of (i) stress response and protein modification and folding, (ii) energy metabolism, (iii) cellular transport and structure, and (iv) nucleic acid binding. In order to not limit the functional grouping to the top 25 listed proteins, GO term enrichment was performed on the entire dataset of MEF and MEF-3 binding proteins. GO term enrichment is not yet readily available for neglected parasites like cestodes, but could be adapted from publicly available enrichment tools for the helminth C. elegans. By this approach highly significant GO hits reflected a very similar grouping as the top 25 proteins. Highly interestingly, many proteins were involved in energy metabolism (GO terms: ATP synthesis coupled electron transport, respirasome, NADH dehydrogenase complex). This is in line with previous studies that found the energy metabolism of Schistosomes, Streptococcus and mammalian cells to be affected by MEF (Martín-Galiano et al., 2002;Manneck et al., 2012;Li et al., 2017;McCann et al., 2018). An important consideration is that binding to a drug does not necessarily lead to functional inhibition. Therefore, follow-up experiments will be needed to prove a functional inhibition of the energy metabolism by MEF.
The binding of proteins involved in cellular transport and structure is also in line with previous observations made in the in vivo study by Küster et al. (2015), where MEF treatment of E. multilocularis metacestodes led to destructive effect on microtriches, which are essential for microtubule-dependent vesicle transport into the laminated layer (Küster et al., 2011).
Stress response and protein modification and nucleic acid binding were further terms the top 25 proteins and GO enrichment of all MEF and MEF-3 specific binding proteins covered. Whereas MEF induces reactive oxygen species and cell death in target cells, it has, to the best of our knowledge, not been shown that specific cell death proteins, proteins of protein modification or folding, nor stress response or nucleic acid, are bound by MEF . They might be highly upregulated in response to MEF treatment, and thereby non-specifically bound to MEF, or MEF could bind central stress response proteins actively and thereby further weaken the parasite. Targeted experiments in the future will show, if MEF also directly affects stress response pathways in the parasite.
In the study by Küster et al. (2015), where a limited and biased proteomic approach based on in-gel-digestion of protein bands was used to identify potential drug interaction partners of MEF, ferritin and cystatin were identified. In other parasites, MEF probably inhibits β-haematin formation during hemoglobin degradation in Plasmodia (Egan et al., 1994;Corrêa Soares et al., 2009) or, respectively, glycolytic enolase in Schistosomes (Manneck et al., 2012). Therefore, it was postulated that MEF might bind metalloproteins like ferritin, haematin or enolase via its strongly electronegative trifluoromethyl group that could interact with metals bound by these metalloproteins. This hypothesis is further backed up by the GO term metal cluster binding that was identified highly enriched amongst the MEF and MEF-3 binding proteins. Further, in our previous SAR study with MEF derivatives it was shown that 4-aminoquinoline derivatives with a trifluoromethyl group in position eight were necessary for in vitro activity against metacestodes (Rufener et al., 2018). In the current more comprehensive DAC study with 14 additional MEF derivatives, ferritin was not ranked within the top 25 binding proteins, but was still included in the repertoire of proteins bound by MEF exclusively. One explanation for this could be the different elution procedure in the previous study, where elution was performed with MEF to attract bound proteins (i.e. specific elution). This may have resulted in a bias towards metalloprotein abundance in the eluates and would also explain why no cystatin was detected in the present study as to compared to the previously performed study (Küster et al., 2015). Further, we could show that ferritin was only bound by MEF and not shared by biologically active MEF-3 and other MEF derivatives. This might explain why MEF showed superior activity against E. multilocularis metacestodes, and might be an additional confirmation of ferritin being a central protein in the activity of MEF against this tapeworm. Future studies will have to address the direct binding of the intracellular iron-storage protein ferritin by MEF.
Also past studies outside the field of Echinococcus have shown that MEF targets multiple pathways: In Plasmodium, it blocks protein synthesis by targeting the cytosolic 80S ribosome, it affects lipid-binding proteins, it inactivates the enzyme metacapsase-1, and it inhibits heme polymerization. Further, MEF was shown to act also on cancer and bacterial cells, and in humans, it was shown to induce hepatic, gastrointestinal, as well as neuropsychiatric side effects. These are mediated as well through multiple pathways, ranging from membrane disorganization, imbalance of small molecule transmembrane transport, deregulated lipid metabolism, destabilization of organelle homeostasis, interruption of signaling pathways, including various described protein targets, yet still the detailed causal mechanisms remain unclear .

Conclusions
The aim of this study was to assess the effecicacy of MEF derivatives against E. multilocularis metacestodes and evaluate their interaction with its proteome to better understand the mode of action of MEF. Based on the drug efficacy assays it can be concluded that none of the derivatives were more effective than MEF against E. multilocularis metacestodes. MEF-16 was one of the most potent among the derivatives. Its structure suggests that, in addition to the trifluoromethyl group, the length of the second residue with similar functional groups to the 2-piperidylmethanol in MEF is important. Using DAC followed by nLC-MS/MS, proteins binding to MEF and MEF-3, but not to ineffective MEF derivatives, were identified. The majority of these binding proteinsthus potential targets -are involved in energy metabolism and cellular transport and structure, and possibly other pathways as well thereby explaining the broad spectrum of MEF against various pathogens and its side effects on hosts.